Improved Phenoxyalkylbenzimidazoles with Activity against

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Improved phenoxyalkylbenzimidazoles with activity against Mycobacterium tuberculosis appear to target QcrB N. Susantha Chandrasekara, Bryan J berube, Gauri Shetye, Somsundaram Chettiar, Theresa O'Malley, Alyssa Manning, Lindsay Flint, Divya Awasthi, Thomas R Ioerger, James C. Sacchettini, Thierry Masquelin, Philip A. Hipskind, Joshua Odingo, and Tanya Parish ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00112 • Publication Date (Web): 16 Oct 2017 Downloaded from http://pubs.acs.org on October 17, 2017

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ACS Infectious Diseases

Improved phenoxyalkylbenzimidazoles with activity against Mycobacterium tuberculosis appear to target QcrB

a

N. Susantha Chandrasekera, aBryan J. Berube, aGauri Shetye, a Somsundaram Chettiar, aTheresa

O’Malley, aAlyssa Manning, aLindsay Flint, aDivya Awasthi, bThomas R. Ioerger, bJames Sacchettini, cThierry Masquelin, c Philip A. Hipskind, a Joshua Odingo, and aTanya Parish*

a

TB Discovery Research, Infectious Disease Research Institute, 1616 Eastlake Avenue E,

Seattle, WA 98102, USA. b

Departments of Computer Science and Engineering and cBiochemistry and Biophysics, 301

Tarrow Street, Texas A&M University, College Station, TX 77843, USA. c

Lilly Research Laboratories, 307 E Merrill St, Indianapolis, IN 46285, USA.

*Corresponding author TB Discovery Research, Infectious Disease Research Institute, 1616 Eastlake Avenue E, Seattle, WA 98102, USA. Tel 206 858 6074 Fax 206 381 3678 Email [email protected]

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ACS Paragon Plus Environment


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The phenoxy alkyl benzimidazoles (PABs) have good anti-tubercular activity. We expanded our structure-activity relationship studies to determine the core components of PABs required for activity. The most potent compounds had minimum inhibitory concentrations against M. tuberculosis in the low nanomolar range with very little cytotoxicity against eukaryotic cells, as well as activity against intracellular bacteria. We isolated resistant mutants against PAB compounds, which had mutations in either Rv1339, of unknown function, or qcrB, a component of the cytochrome bc1 oxidase of the electron transport chain. QcrB mutant strains were resistant to all PAB compounds, whereas Rv1339 mutant strains were only resistant to a subset, suggesting that QcrB is the target. The discovery of the target for PAB compounds will allow for the improved design of novel compounds to target intracellular M. tuberculosis.

Key Words: Tuberculosis, benzimidazole, respiratory inhibitors, drugs

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Mycobacterium tuberculosis was a leading cause of death by infectious disease in the world in 2015 1. Contributing to over 1.4 million deaths and infecting an estimated one-third of the population, M. tuberculosis is a leading global health problem with major social and economic burdens 1. Over the last decade, there has also been an alarming increase in multidrugresistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains that are refractory to conventional therapeutics 1. Because of this, there is a dire need for novel drugs and preventatives to combat this disease. The last few years have seen promising leads for new anti-tuberculosis drugs with several advancing to clinical trials. One series in the early stages of development, the phenoxy alkyl benzimidazoles (PAB), has shown good promise with minimum inhibitory concentrations (MICs) against M. tuberculosis in the nanomolar range, sterilizing activity under starvation conditions, and low cytotoxicity against eukaryotic cells 2. In this study, we further characterized the SAR for the PAB series and synthesized compounds with anti-tubercular activity in the low nanomolar range, good cytotoxicity profiles, and intracellular activity against M. tuberculosis. We also identified the probable target of PAB compounds as QcrB – a component of the cytochrome bc1 oxidase of the electron transport chain. This knowledge will enable the elucidation of the mechanism of action for the strategic design of new compounds and combination therapies to treat M. tuberculosis infection.

RESULTS AND DISCUSSION PAB Structure-Activity Relationship

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The alkyl benzimidazoles were synthesized according to Scheme 1. Condensation of appropriate 1,2-diaminobenzene derivative with propionic acid yielded the benzimidazole intermediate (1) upon heating.

Alkylation of the benzimidazole intermediate (1) was

accomplished by reacting with dibromoalkane to form N-(bromoalkyl)benzimidazole (2). This in turn was treated with the corresponding anilines, thiophenols and phenols to yield the corresponding benzimidazole alkylamine, alkyl thioether and alkyl ethers.

Scheme 1: Synthesis of amine and thioether aryl, hetero aryl, cycloalkyl benzimidazoles.

In our previous study, we observed that compounds with heteroatoms (O, S, and N) as part of the linker were tolerated, a linker length of three to four carbons was favored, and ethyl at the 2position and methyl at the 6-position on the benzimidazole were the most favorable substituents on the core 2. We first investigated aryl and heteroaryl thioethers for their activity against M. tuberculosis. Since compound 4 from our previous work showed good activity (Table 1) 2, we made analogs (6 – 18) to explore the SAR around the phenyl group of 4. In our previous studies on aryl ethers, we observed that the 4-methyl substitution improved both activity and selectivity. In the case of the aryl thioether analogs, 4-methyl substitution (6) did not show any improvement in activity, while 4-methoxy substitution (7) decreased activity by 4-fold (MIC = 4.8 µM). 4


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Replacement of the phenyl with biaryls or a heteroaryl ring, such as naphthalene (8), quinoline (9), quinazoline (10), benzothiazole (11 & 12) or benzimidazole (13 & 14) decreased activity while replacement with thiazole (15 & 16) or tetrazole (17) resulted in no activity (MIC >20 µM). The 4-phenylthiazole (18) was the most active thioether analog with an MIC of 0.73 µM. However, it was fairly cytotoxic to eukaryotic cells (TC50 = 9.7 µM), despite the selectivity index (SI = 13) being similar to the earlier compounds.

Table 1: Effect of phenyl replacements on biological activity `

N

N

N

R

N

3-5

SR 6-18

Compound

Name

R-group

MIC99a (µM)

TC50b (µM)

SIc

3d

IDR-0333819

Phenoxy

1.1 ± 0.4

21 ± 7.1

19

4d

IDR-0341914

Phenylthio

1.4 ± 0.3

31 ± 8.3

22

d

IDR-0351544

Anilinyl

0.47 ± 0.09

42 ± 7.8

89

6

IDR-0357446

4-Methylphenyl

1.4 ± 0.56

19 ± 0.71

14

7

IDR-0458791

4-Methoxyphenyl

4.8 ± 0.1

34 ± 12.8

7

8

IDR-0357447

2-Naphthyl

4.1 ± 1.3

10 ± 0

2

9

IDR-0357452

2-Quinolinyl

18 ± 2.6

7±0

0.4

10

IDR-0357451

2-Quinoxalinyl

10 ± 0

13 ± 0

1

11

IDR-0351547

6-Ethoxy-2-benzothiazolyl

10 ± 2.1

12 ± 0.7

1

12

IDR-0351549

6-Chloro-2-benzothiazolyl

6.0 ± 4.4

18 ± 2.2

3

13

IDR-0351556

2-Benzimidazolyl

>20

18 ± 2.8

NC

14

IDR-0351555

N-methyl-2-benzimidazolyl

>20

16 ± 2.1

NC

15

IDR-0351552

2-Thiazolyl

>20

>50

NC

16

IDR-0351550

5-Methyl-2-thiazolyl

>20

>50

NC

17

IDR-0351557

N-Methyl-5-tetrazolyl

>20

>50

NC

18

IDR-0351553

4-Phenyl-2-thiazolyl

0.73

9.7

13

5

5



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a

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MIC99 is the minimum concentration required to completely inhibit growth of M. tuberculosis in liquid culture.

MICs of active compounds are the average of two independent experiments ± standard deviation. b TC50 is the concentration required to inhibit growth of Vero cells by 50%. TC50 is the average of two independent experiments ± standard deviation. c Selectivity index (SI) is TC50/MIC. NC - not calculated. d Data for compounds 3, 4, 5 are from compounds 6, 53, 54, respectively in 2.

In our previous SAR study, we observed that replacing the phenyl ether in compound 3 with 5(4-chlorophenyl)oxadiazol-2-yl thioether as in 19 improved activity 2. We attempted to improve this further by modifying the linker length (Table 2). Reduction of the linker length from four carbons to three (20) or two (21) was detrimental to the anti-tubercular activity; in the case of the propyl (20) linker, we observed a 39-fold reduction in activity and further reduction of the linker to an ethyl (21) resulted in complete loss of activity. The addition of a weaker electron-releasing group such as the methyl group on the 6-position of the benzimidazole core (22) retained good potency (MIC = 0.085 µM). The combination of a 6-methyl on the benzimidazole core and a propyl linker (23) was slightly less active in comparison (MIC =0.34 µM). Interestingly, replacing the 6-methyl with 6-methoxy, a strong electron-donating group, in compound 24, reduced activity by almost 10-fold (MIC = 2.5 µM).

Table 2: Effect of N-alkyl chain length and benzo substitution on biological activity. Compound

Name

19d

IDR-0351551

Structure

6


MIC99a (µM)

TC50b (µM)

SIc

0.10 ± 0.07

22 ± 9.2

220

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a

20

IDR-0390229

3.9 ± 0.21

21 ± 2.1

5

21

IDR-0351587

>20

35 ± 2.8

NC

22

IDR-0390228

0.085 ± 0.007

18 ± 0

212

23

IDR-0390226

0.34 ± 0.19

21 ± 2.8

62

24

IDR-0458783

2.5 ± 0.2

49 ± 2.6

20


MICs of active compounds are the average of two independent experiments ± standard deviation.

b

TC50 is the

concentration required to inhibit growth of Vero cells by 50%. TC50 is the average of two independent experiments ± standard deviation. c Selectivity index (SI) is TC50/MIC. NC - not calculated.

d

Data for compound 19 is from

compound 68 in 2.

We next explored the influence of nitrogen as the heteroatom in the linker (Table 3). We synthesized compounds (25 - 35) to investigate the electronic and steric effects of different aromatic substituents on the terminal aniline. The addition of the electron donating groups methyl (25) or methoxy (26), made no improvement in activity, although the methyl analog was slightly more active. A trifluoromethyl substituent at the meta position (27) had a comparable MIC to compound 5. However, the cytotoxicity was significantly worse than that of 5 and the selectivity was 8-fold lower. The 3,5-dimethoxy (28) lost activity, whereas the 3-halo and 4-

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methyl substituted anilines (29,30) showed comparable activity to that of 5. The substituents 3benzyloxy (31) and 4-pyridyl (32) showed similar activity to that of 5, but the 3-pyridyl (33) was much less active (MIC = 3.7 µM). Moving from the aniline (5) to benzylamine (34) was detrimental to the anti-tubercular activity. Replacing the terminal aniline with a morpholino group also resulted in loss of activity (35). Table 3: Effect of anilinyl substitution on the biological activity.

a

Compound

Name

R

MIC99a (µM)

TC50b (µM)

SIc

5d

IDR-0351544

Anilinyl

0.47 ± 0.09

42 ± 7.8

89

25

IDR-0357431

4-Methylanilinyl

0.50 ± 0.1

39 ± 1.4

78

26

IDR-0458786

4-Methoxyanilinyl

1.4 ± 1.1

>100

>71

27

IDR-0351602

3-Trifluoromethylanilinyl

1.1 ± 0.9

12 ± 2.6

11

28

IDR-0351605

3,5-Dimethoxyanilinyl

4.4 ± 1.5

21 ± 4.6

5

29

IDR-0351599

3-Bromo-4-methylanilinyl

0.45 ± 0.07

17 ± 0

38

30

IDR-0458790

3-Chloro-4-methylanilinyl

0.35 ± 0.07

53 ± 27.0

151

31

IDR-0351600

3-Benzyloxyanilinyl

0.60 ± 0

14 ± 3.8

23

32

IDR-0351601

4-(2-Pyridyl)anilinyl

0.80 ± 0.7

22 ± 2.1

28

33

IDR-0351606

3-(2-Pyridyl)anilinyl

3.7 ± 0.8

19 ± 3.8

5

34

IDR-0351603

N-Benzylamino

>20

>50

NC

35

IDR-0357457

N-Morpholinyl

>20

>100

NC



b

TC50 is the

concentration required to inhibit growth of Vero cells by 50%. TC50 is the average of two independent experiments ± standard deviation.

c

Selectivity index (SI) is TC50/MIC. NC - not calculated.

compound 54 in 2.

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d

Data for compound 5 is from

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4-Bromo-N-(3-(2-ethyl-6-methyl-1H-benzo[d]imidazol-1-yl)propyl)-3-methylaniline, compound 62 from Chandrasekera et al.

2

was the most potent and selective in our previous study 2. This

compound contained only three carbons in the lipophilic linker. We prepared additional analogs 36 - 43 to understand the SAR surrounding the phenyl region of three-carbon tethered benzimidazole molecules. The phenyl ether analog (36) had good activity (MIC = 5.2 µM) but the corresponding aniline analog (37) was 10-fold more active. Electron withdrawing 4-methyl substitution on phenyl ether (38) or thioether (39) analogs improved activity. The aniline analog with a 4-methyl group (40) also showed good activity (MIC = 0.12 µM) and selectivity (SI = 258). In comparison, double substitution to the more electron-rich 3,4-dimethylaniline analog (41) slightly reduced activity but a combination of electron-withdrawing group at 3-position and methyl at 4-position (42) resulted in the most potent of all the alkyl benzimidazoles (Table 4) with a MIC of 0.040 µM and good selectivity (SI = 275). Interestingly, a 3-chlorine substitution instead of the 3-bromine substitution was ~10-fold less active (MIC = 0.55 µM).

Table 4: Effect of phenyl ring substitutions on biological activity.

Compound

Name

R

MIC99a (µM)

TC50b (µM)

SIc

36d

IDR-0257287

Phenoxy

5.2 ± 1.9

>50

>10

37

IDR-0357449

Anilinyl

0.45 ± 0.2

29 ± 4.9

64

38

IDR-0357444

4-Methylphenoxy

1.6 ± 0.2

14 ± 0.7

9

39

IDR-0357448

4-Methylphenylthio

4.5 ± 0.8

24 ± 2.8

5

9



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a

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40

IDR-0357450

4-Methylanilinyl

0.12 ± 0.07

31 ± 3.5

258

41

IDR-0357442

3,4-Dimethylanilinyl

0.65 ± 0.4

>100

>154

42

IDR-0357458

3-Bromo-4-methylanilinyl

0.04 ± 0

11 ± 0.7

275

43

IDR-0357443

3-Chloro-4-methylphenoxy

0.55 ± 0.35

16 ± 0

29



b

TC50 is the

concentration required to inhibit growth of Vero cells by 50%. TC50 is the average of two independent experiments ± standard deviation. c Selectivity index (SI) is TC50/MIC. d Data for compound 36 is from compound 5 in 2.

We then investigated the effect of 6-methyl substitution on the benzimidazole core bearing a 4carbon tether to various aromatics (Table 5). The aniline (44), phenyl thioether (45) and phenyl ether (46) had comparable activities. 4-methoxy substitution on phenyl ether (47) or aniline (48) had minimal effect on activity, but this substitution resulted in 10-fold shift in activity of a corresponding phenyl thioether (49). Both 3-chloroaniline (50) and 4-chlorophenyl thioether (53) were less active but a 3-trifluoromethyl substitution on aniline (51) retained all activity of parent compound 44. We also tested 3-benzyloxy aniline (52) with good activity.

Table 5: Effect of phenyl SAR on biological activity.

Compound

Name

R

MIC99a (µM)

TC50b (µM)

SIc

44d

IDR-0357433

Anilinyl

0.32 ± 0.09

22 ± 0.7

69

45

IDR-0341934

Phenylthio

0.22 ± 0.02

9.5 ± 0.7

43

10


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a

46d

IDR-0341930

Phenoxy

0.27 ± 0.09

18 ± 2.5

67

47

IDR-0458750

4-Methoxyphenoxy

0.4 ± 0.1

27 ± 6.4

68

48

IDR-0458784

4-Methoxyanilinyl

0.65 ± 0.07

14 ± 2.1

22

49

IDR-0458789

4-Methoxyphenylthio

2.3 ± 0.4

20 ± 2.1

9

50

IDR-0357439

3-Chloroanilinyl

2.1 ± 0.9

16 ± 9.0

8

51

IDR-0357436

3-Trifluoromethylanilinyl

0.55 ± 0.4

5.7 ± 1.5

10

52

IDR-0357435

3-Benzyloxyanilinyl

1.8 ± 0.76

18 ± 0.7

10

53

IDR-0357445

4-Chlorophenylthio

2.6 ± 0.6

16 ± 1.4

6

54

IDR-0578347

Anilinyl

0.056 ± 0.020

> 50

>893

55

IDR-0390234

4-Methylanilinyl

0.066 ± 0.018

31 ± 0.7

470

56

IDR-0458788

4-Methoxyanilinyl

0.35 ± 0.2

91 ± 12.3

260

57

IDR-0390235

2,4-Dimethylanilinyl

0.070 ± 0.03

14 ± 0.7

200

58

IDR-0458745

4-Methoxyphenylthio

0.88 ± 0.2

33 ± 11

38


MICs of active compounds are the average of two independent experiments ± standard deviation. b TC50 is the concentration required to inhibit growth of Vero cells by 50%. TC50 is the average of two independent experiments ± standard deviation. c Selectivity index (SI) is TC50/MIC. d Data for compounds 44 and 46 are from compound 55 and 34, respectively in 2.

We finally probed the effect of combination of the optimized residues, namely the three-carbon linker and a 6-methyl substitution on the benzimidazole core (54 – 58). Four compounds had a selectivity index of > 200 (54, 55, 56 & 57) (Table 5). The aniline analogs 54, 55 and 57 had the best activities with comparable MICs of 0.061 µM, 0.067 µM and 0.070 µM, respectively. While 54 had no cytotoxicity at 50 µM, the addition of one (55) or two (56) methyl groups on the phenyl ring caused a step-wise increase in cytotoxicity towards eukaryotic cells, thus reducing the selectivity index from > 819 (54) to 470 and 200 for 55 and 57, respectively (Table 5). The methoxy analog (56) was 5-fold less active (Table 5).

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Several phenoxy alkyl imidazoles 59 – 68 were evaluated to study the importance of the benzimidazole core for the anti-tubercular activity of PAB (Table 6). Many of the compounds lost activity (59, 60, 64 & 67), while others maintained only moderate activity compared to benzimidazole variants (61, 62, 63, 65, 66 & 68). These data show the benzene of the benzimidazole core is not strictly required; several imidazole variants maintain moderate activity against M. tuberculosis. However, the benzene ring does appear to confer tubercular activity to PAB compounds.

Table 6: SAR of phenoxy alkyl imidazole analogs with varying linker length.

Compound

Name

MIC99a (µM)

TC50b (µM)

SIc

59

IDR-0122293

> 20

> 50

NC

60

IDR-0124213

> 20

ND

NC

61

IDR-0124480

20

16 ± 0

0.8

62

IDR-0257359

11 ± 2.3

35 ± 11

3

63

IDR-0262047

20

21

1

64

IDR-0262049

> 20

ND

NC

65

IDR-0262053

20

40

2

66

IDR-0155469

9.9 ± 1.7

19 ± 9.3

2

12


optimal anti-

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a

67

IDR-0123367

> 20

89 ± 2.5

NC

68

IDR-0168354

8.6 ± 0.9

15 ± 0.3

2



b

TC50 is the

concentration required to inhibit growth of Vero cells by 50%. TC50 is the average of two independent experiments ± standard deviation. c Selectivity index (SI) is TC50/MIC. ND – not determined. NC - not calculated.

PAB compounds target QcrB To identify the target of PAB compounds, we isolated resistant mutants against three different PABs. Of the seven confirmed mutants, five contained mutations in qcrB, the cytochrome b subunit of the cytochrome bc1 oxidase (Rv2196), while two of the three mutants raised against compound 4 contained mutations in Rv1339, a gene of unknown function (Table 7).

Table 7: Resistant mutant strains isolated against PAB compounds. Compound

Name

Structure

Gene

Mutation

qcrB

A179P

N

3

IDR-0333819

N O

N

4

IDR-0341914

N S

19

IDR-0351551

13


Rv1339

V90M

qcrB

A179P

Rv1339

Y94C

qcrB

M342T

qcrB

W312C

qcrB

W312G


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We confirmed targeting of QcrB by PABs by testing the MIC of 30 active compounds against LP-0351551-RM1 (QcrBM342T), one of the isolated resistant mutants. All compounds tested showed a minimum 5-fold shift in MIC compared to the parental strain with 78% of the compounds having at least a 10-fold higher MIC (Table 8). Most compounds had similar MICs against LP-0341914-RM5 (Rv1339V90M) as against the wild-type strain (Table 9). Six compounds had 5-fold shifts in MIC against the Rv1339V90M mutant, but in all cases the observed resistance shift was significantly smaller than those observed when the same compounds were tested against the QcrBM342T mutant (Tables 8 + 9). Together, these data indicate the Rv1339 gene product is unlikely to play a major role in resistance to the PAB series and implicates QcrB as the target of PAB compounds.

Table 8: Cross-resistance of QcrBM342T mutant strain to PAB compounds. Compound

Name

WT MIC90a (µM)

QcrBM342T MIC90a (µM)

Foldchange

3

IDR-0333819

16

>100

>6.3

4

IDR-0341914

20

>100

>5.0

6

IDR-0357446

8.3

>100

>12

18

IDR-0351553

5.7

>100

>18

19

IDR-0351551

0.15

47

313

20

IDR-0390229

8

>100

>13

22

IDR-0390228

6

>100

>17

26

IDR-0458786

6.9

>100

>15

28

IDR-0351605

1

5

5.0

30

IDR-0458790

1.6

93

58

32

IDR-0351601

0.55

22

40

37

IDR-0357449

2.7

>100

>37

38

IDR-0357444

7.6

>100

>13

41

IDR-0357442

3.1

35

11

14


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a

43

IDR-0357443

3.8

44

12

44

IDR-0357433

0.21

8.6

41

46

IDR-0341930

1.3

73

56

49

IDR-0458789

9.4

98

10

52

IDR-0357435

6.7

70

10

53

IDR-0357445

16

>100

>6.3

54

IDR-0578347

0.10

4.1

41

57

IDR-0390235

1

6.2

6.2

58

IDR-0458745

4.6

98

21

69

b

IDR-0341919

0.92

16

17

70

b

IDR-0341920

5

>100

>20

71

b

IDR-0341931

7.7

87

11

72b

IDR-0341936

14

>100

>7.1

73b

IDR-0341926

3.5

31

8.9

74

b

IDR-0341921

13

>100

>7.7

75

b

IDR-0357430

0.88

48

55

76

b

IDR-0390233

0.66

>100

>152

MIC90 is the concentration required to inhibit growth of M. tuberculosis by 90%. LP-0351551-

RM1 is QcrBM342T.

b

Compounds 69-76 are compounds 13, 15, 22, 23, 29, 30, 56, 62,

respectively in 2.

Table 9: Cross-resistance of M. tuberculosis Rv1339V90M mutant strain to PAB compounds. Compound

Name

WT MIC90a (µM)

Rv1339V90M MIC90a (µM)

Foldchange

3

IDR-0333819

16

6.2

0.4

18

IDR-0351553

5.7

9.6

1.7

19

IDR-0351551

0.15

1.5

10

20

IDR-0390229

8

10

1.3

22

IDR-0390228

6

2.9

0.5

26

IDR-0458786

6.9

3

0.4

30

IDR-0458790

1.6

0.77

0.5

32

IDR-0351601

0.55

6.3

12

37

IDR-0357449

2.7

1.8

0.7

38

IDR-0357444

7.6

6.9

0.9

46

IDR-0341930

1.3

1.7

1.3

15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

49

IDR-0458789

9.4

10

1.1

52

IDR-0357435

6.7

3.5

0.5

53

IDR-0357445

16

12

0.8

58

IDR-0458745

4.6

6.8

1.5

b

IDR-0341919

0.92

6.2

6.7

70b

IDR-0341920

5

2.3

0.5

71

b

IDR-0341931

7.7

5.9

0.8

72

b

IDR-0341936

14

13

0.9

73

b

IDR-0341926

3.5

2.1

0.6

74

b

IDR-0341921

13

7.8

0.6

75

b

IDR-0357430

0.88

0.74

0.8

76

b

IDR-0390233

0.66

0.62

0.9

69

a

Page 16 of 64

MIC90 is the concentration required to inhibit growth of M. tuberculosis by 90%. b Compounds 69, 70, 71, 72, 73,

74, 75, 76 are compounds 13, 15, 22, 23, 29, 30, 56, 62, respectively from 2.

Structurally, the benzene of the benzimidazole core of the PAB series is not required for antituberculosis activity, as some compounds with just an imidazole ring (PAB-II) still have moderate activity against M. tuberculosis (Table 6). However, the PAB-II compounds appear to have a different target or mode of action, since the QcrBM342T mutant strain was not resistant to this set (Table 10). This argues that while the benzene ring of the core may not be strictly required for whole-cell activity, this ring may be responsible for targeting of QcrB specifically. It would be interesting to elucidate the target of PAB-II compounds to determine their mechanism of action.

Table 10: Cross-resistance of M. tuberculosis QcrBM342T mutant strain to PAB-II compounds. Compound

Name

WT MIC90a

QcrBM342T MIC90a (µM)

Foldchange

16


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(µM)

a

63

IDR-0262047

34

47

1.4

66

IDR-0155469

8.1

8.7

1.1

68

IDR-0168354

9.4

10

1.1

MIC90 is the concentration required to inhibit growth of M. tuberculosis by 90%.

As a key component of the mycobacterial electron-transport chain, QcrB receives electrons from menaquinol and serves as a pump shuttling protons into the periplasmic space, thereby establishing the proton gradient required for ATP production 3. Over the last few years, the cytochrome bc1 oxidase has been discovered to be the target of novel drugs against numerous infectious diseases including malaria and Leishmania parasites, as well as multiple classes of compounds active against M. tuberculosis, including the drug Q203, an imidazopyridine amide currently in clinical trials

4-8

. Two of the resistance mutations found in our study cause amino

acid changes at position 312 of QcrB, which sits in the same pocket as the resistant mutant at position 313 found for Q203 8. This suggests the PAB compounds may be binding in the same location and have a similar mechanism of action as Q203. To test this, we examined crossresistance of PAB compounds to a strain of M. tuberculosis containing the same mutation found in Q203-resistant isolates (QcrBT313I). PAB compounds were highly resistant to the QcrBT313I variant; the MIC90 for PAB compounds ranged from 13-fold up to 200-fold higher compared to the wild-type strain (Table 11). The fold-change in MIC against QcrBT313I was larger than that for QcrBM342T for every compound tested except IDR-0351551, the compound against which QcrBM342T was raised. When Q203 was tested against QcrBW312C, the MIC was greater than 100

17



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Page 18 of 64

nM compared to just 2.8 nM for H37Rv-LP. Together, these data suggest an overlapping binding site between Q203 and PAB compounds.

Table 11: Cross-resistance of QcrBT313I mutant strain to PAB compounds. Compound

Name

WT MIC90a (µM)

QcrBT313I MIC90a (µM)

Foldchange

19

IDR-0351551

0.39

63

161

28

IDR-0351605

2.4

> 100

> 41

32

IDR-0351601

1.9

> 100

> 52

44

IDR-0357433

0.45

89

198

52

IDR-0357435

2.2

> 100

> 45

54

IDR-0578347

0.10

8.7

87

69

b

IDR-0341919

0.87

61

70

71

b

IDR-0341931

7.7

> 100

> 13

77

b

Q203

0.0088

1.7

193

a

MIC90 is the concentration required to inhibit growth of M. tuberculosis by 90%. b Compounds

69 and 71 are compounds 13 and 22, respectively in 2. Compound 77 is from 8.

To provide further evidence supporting QcrB as the target of PAB compounds, we performed initial mechanistic studies surrounding QcrB. Inhibition of QcrB leads to reduced proton pumping across the bacterial membrane. An inability to pump protons disrupts ATP production and increases the concentration of protons within the cell, thus lowering intrabacterial pH (pHIB). As would be expected following QcrB inhibition, PAB compounds caused ATP depletion over a 24 hr time-period (Fig. 1A), and significantly decreased pHIB; all PAB compounds caused a reduction in pHIB to at least 6.7 with 8/16 compounds dropping the pHIB below 6.5, similar to that seen with the ionophore monensin (Figure 1B). The one PAB-II 18


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compound tested showed no activity in this assay, consistent with the conclusion QcrB is not its target. Together with the resistant mutant isolation, these data strongly support QcrB as the target for PAB compounds and suggest potential mechanisms for compound activity.

Previous studies identified PAB compounds as bacteriostatic against actively growing M. tuberculosis, while bactericidal against nutrient-starved bacteria 2. We tested this for two compounds, including one of our most potent compounds, IDR-0578347 (54). Both compounds had similar activity; when tested at 10X their respective MICs, the compounds inhibited growth of M. tuberculosis grown aerobically, but displayed bactericidal activity only in the context of nutrient starvation (Fig. 2A and 2B). One possible explanation for the differential activity stems from the elucidation of QcrB as the target. Under replicating conditions individual bacteria contain large amounts of ATP necessary to support growth and division of bacterial cells. Under starvation conditions ATP is still required to maintain basal cellular processes, but the amount of ATP/cell is much reduced 9. It is possible ATP depletion mediated by PAB compounds during replicative growth is only enough to inhibit growth and force the bacteria into a non-replicative state, while under starvation conditions any reduction from the basal amount of ATP being produced is enough to cause bacterial cell death. This hypothesis still needs to be tested experimentally.

The number of series of compounds that target QcrB has led to an intriguing debate within the field. On one hand, the number of times this target has been identified highlights the importance of the cytochrome bc1 complex and ETC to mycobacterial survival 3. However, there has been 19



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Page 20 of 64

concern as to the promiscuity of the target, the similarity to the human electron transport chain, and the ability of M. tuberculosis to adapt by using the bd oxidase as an alternative electron acceptor under oxygen-limited conditions

10-11

. Recent work used the adaptability of M.

tuberculosis against itself by using combination therapy targeting the ETC

12

. Based on our

results, it is possible the PAB series could similarly be used in combination with other electrontransport inhibitors. These studies are currently underway.

Table 12: Activity of PAB compounds against intracellular M. tuberculosis. Compound

ID

Intracellular IC50a (µM)

Intracellular IC90b (µM)

TC50 (µ µM)c

SId

18

IDR-0351553

0.27 ± 0.05

0.86 ± 0.14

17 ± 3.5

19

44

IDR-0357433

0.040 ± 0.002

0.15 ± 0.04

49 ± 14.9

325

46

IDR-0341930

0.070 ± 0.006

0.29 ± 0.06

28 ± 0.5

95

54

IDR-0578347

0.0073 ± 0.0009

0.028 ± 0.001

>50

>1785

73

IDR-0341926

0.093 ± 0.01

0.30 ± 0.05

26 ± 3.2

85

e

IDR-0341939

0.48 ± 0.03

1.3 ± 0.16

38 ± 5.6

29

77

a

Intracellular IC50 is the concentration required to inhibit growth of M. tuberculosis inside macrophages by 50%.

b

Intracellular IC90 is the concentration required to inhibit growth of M. tuberculosis inside macrophages by 90%.

c

TC50 is the concentration required to inhibit growth of RAW cells by 50%. Data are the average of four experiments

± standard deviation. d Selectivity index (SI) = TC50/IC90. e Compound 77 is compound 67 from 2.

As for the potential consequence of targeting a component of the electron transport chain, our previously published data suggests the inhibitors described herein are specific to M. tuberculosis, as PAB compounds do not inhibit growth of several other bacteria, including M. smegmatis 2. In fact, when tested against M. tuberculosis in a high-content analysis of M. tuberculosis macrophage infection, PAB compounds showed exquisite sensitivity towards bacterial cells with 20


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MICs against M. tuberculosis inside macrophages similar to those found in liquid broth and selectivity indexes ranging from 19 to nearly 2000 (Table 12). Several compounds even appeared to be slightly more active inside macrophages than in liquid culture. Together, these data show the penetrance of these compounds into eukaryotic cells, while highlighting the safety of the PAB compounds, offering a promising therapeutic window that would allow for treatment of infection while minimizing host cell toxicity.

CONCLUSION This work investigated a range of possible amine and thioether linkages as a strategy to identify improved alkyl benzimidazole molecules. Several compounds showed good potency and selectivity, as well as activity against intracellular bacteria. The key determinants for optimal antimycobacterial activity of the compounds are methyl groups in both the C-6 position of the benzimidazole and the para position of the terminal phenyl ring, a three or four carbon atom linker, a nitrogen as the hetero atom on the alkyl chain, and a benzimidazole as the core moiety. The PAB compounds appear to target QcrB, a component of the M. tuberculosis electron transport chain, suggesting they could be useful in combination therapy with other electrontransport chain inhibitors.

METHODS Bacterial strains and growth conditions M. tuberculosis H37Rv (London Pride) (ATCC 25618) 13 was used for all work. M. tuberculosis strains were grown in Middlebrook 7H9 medium containing 10% v/v OADC (oleic acid, 21



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Page 22 of 64

albumin, dextrose, catalase) supplement (Becton Dickinson) and 0.05% w/v Tween 80 (7H9-TwOADC) under aerobic conditions. Determination of minimum inhibitory concentration (MIC) MICs were performed as previously described

14

; briefly MIC99s were determined against M.

tuberculosis H37Rv-LP constitutively expressing codon-optimized mCherry grown in 7H9-TwOADC under aerobic conditions. Bacterial growth was measured by OD590 after 5 days of incubation at 37 °C. MIC99 was defined as the minimum concentration required for >99% growth inhibition using the Gompertz model. MIC90s were determined against M. tuberculosis H37RvLP or indicated mutants under the same growth conditions. MIC90 is defined as the concentration of compound required to inhibit growth of M. tuberculosis by 90% and was determined from the Levenberg-Marquardt least-squares plot 15. Cytotoxicity assay Cytotoxicity was determined against the Vero African green monkey kidney cell line (ATCC CCL-81). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM), High Glucose, GlutaMAX™ (Invitrogen), 10% v/v FBS (Fetal Bovine Serum), and 1x of PenicillinStreptomycin Solution (100 units/mL of penicillin, 100 µg/mL of streptomycin). Cells were plated into 96-well plates and allowed to adhere for 24 hours before exposure to compounds for 48 h. CellTiter-Glo® Reagent (Promega) was added to 96-well plates and relative luminescent units (RLU) were measured. Inhibition curves were fitted using the Levenberg–Marquardt algorithm. Toxic concentration (TC50) was defined as the concentration of compound that gave 50% inhibition of growth. Resistant mutant isolation 22


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M. tuberculosis H37Rv-LP was plated on 7H10-OADC plates containing 5X or 10X MIC of three PAB compounds #3, 4, and 19 (Compounds 6, 53, and 68 from 2). Resistant clones raised against compounds 4 and 19 were isolated, confirmed and whole genome-sequenced as previously described

16

. The resistant clone raised against compound 3 was isolated, and

confirmed by sequencing. ATP depletion assay M. tuberculosis H37Rv-LP was grown in duplicate at 37 °C for 24 hrs in the presence of 3-fold dilutions of PAB compounds starting at 200 µM. ATP levels were measured using the BacTiterGlo assay kit (Promega) according to the manufacturer’s instructions. OD590 of the duplicate plate was measured using a Synergy H4 plate reader (BioTek). ATP/OD was calculated by dividing relative fluorescent units from the BacTiter-Glo plate by OD590 readings. pH homeostasis assay M. tuberculosis H37Rv-LP containing a pH-sensitive ratiometric GFP reporter

17

was added to

compounds at 100 µM final concentration in phosphocitrate buffer at pH 4.5. After 48 hrs fluorescence was read and intrabacterial pH was determined based on the ratio between Ex396/Em510 and Ex475/Em510. Determination of compound kill kinetics M. tuberculosis H37Rv-LP was inoculated into 7H9-Tw-OADC at 1 x 105 CFU/mL in the presence of 10X the respective MIC of either compound 46 or 54 or a DMSO control (final DMSO concentration of 2%). For starvation experiments, H37Rv-LP was washed and resuspended in phosphate-buffered saline (PBS) + 0.05% Tyloxapol at 1 x 105 CFU/mL for 2

23



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weeks prior to compound addition. Serial dilutions were performed at the indicated times and bacteria were plated on 7H10-OADC agar plates for CFU enumeration. Intracellular activity Activity against intracellular bacteria was tested as described 18. In brief, RAW 264.7 cells were batch-infected overnight in RPMI-1640, 10% v/v FBS, 2 mM Glutagro, 1 mM sodium pyruvate medium with M. tuberculosis DREAM8 (an H37Rv variant constitutively expressing dsRED) 19 at a multiplicity of infection of 1. Cells were harvested, washed twice and seeded into 384-well assay plates at 3.3 x 103 cells per well. Infected cells were exposed to compounds for 72 hours, Macrophage nuclei were stained with SYBR Green I dye and imaged on an ImageXpress Micro with a 4X objective using a FITC filter set. M. tuberculosis were imaged using a Texas Red filter set. Images were analyzed by setting a lower bound for pixel intensity for each channel to define nuclei and bacteria above background. The integrated intensity of each channel above this threshold was determined. Raw data were normalized to the average integrated control wells. Growth inhibition curves were fitted using the Levenberg–Marquardt algorithm. The IC50 and IC90 were defined as the compound concentration that produced 50% or 90% of the growth inhibitory response, respectively, for each readout.

COMPOUND SYNTHESIS Compounds 59-68 were purchased from Chembridge.

General Methods 1

H NMR (300MHz) spectra were recorded on a Bruker Biospin NMR spectrometer. Reactions

were monitored using thin-layer chromatography (TLC) using Whatman silica gel 60 Å plates 24


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Page 25 of 64

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with florescent indicator and visualized using a UV lamp (254 nm). Flash chromatography was performed on Grace with GraceResolv Normal Phase disposable silica columns. High performance liquid chromatography (HPLC) was performed on a Gilson 322 HPLC pump with a Gilson UV/VIS-155 detector and a Phenomenex Gemini C18 column (10 µm, 250 mm x 10 mm). The purity of all final products was >95% as determined by HPLC analysis conducted on an Agilent 1100 HPLC-MS system (Phenomenex Gemini C18 column, 5 µm, 3 x 50 mm, 0.45 mL/min, UV 254 nm, room temperature) with gradient elution (5-95% acetonitrile in water over 8 min with all solvents containing 0.05% formic acid). Liquid chromatography electrospray ionization mass spectroscopy (LC-MS/ESI-MS) were acquired on an Agilent LC/MSD-SL with a 1100 HPLC and G1956B mass spectrometer with a Phenomenex Gemini 5 µm C18 110Å 50x3 mm column. High-resolution mass spectra (HRMS-ESI) were acquired by the Mass Spectrometry Laboratory at the University of Michigan on an Agilent Q-TOF HPLC-MS.

General procedure for synthesis of 1H-benzo[d]imidazole intermediates (1): 1H-benzo[d]imidazole intermediates were synthesized according to reported procedure.2

General procedure for synthesis of 1-(2-bromoalkyl)-1H-benzo[d]imidazole intermediates (2): 1-(2-bromoalkyl)-1H-benzo[d]imidazole intermediates were synthesized according to reported procedure 2.

25



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Page 26 of 64

For the synthesis of 1-(2-bromoalkyl)-6-methyl-1H-benzo[d]imidazole intermediates, two isomers were formed and separated chromatographically. Both isomers were characterized by 1H NMR and the desired intermediate was carried forward 2.

General procedure for preparation of compounds 6-18, 25-35, 37-43, 45 and 47-58: To a solution of benzimidazole in dimethylformamide were added 5 molar equivalents of potassium carbonate and 2 molar equivalents of the appropriate alcohol (or thiol). The reaction was stirred overnight at room temperature or until disappearance of the starting material monitored by TLC. Heating of the reaction to 50 °C was necessary to prepare the amine analogs using the same protocol. The reaction mixture was washed with water and extracted with ethyl acetate. The organics were dried with anhydrous sodium sulfate and concentrated in vacuo. The crude mixture was purified by column chromatography. General procedure for preparation of compounds 20-24: To a solution of benzimidazole in dimethylformamide were added 5 molar equivalents of potassium carbonate and 2 molar equivalents of 5-(4-chlorophenyl)-1,3,4-oxadiazole-2-thiol. The reaction was stirred overnight at room temperature or until all of the starting material disappeared. The reaction mixture was washed with water and extracted with ethyl acetate. The organics were dried with anhydrous sodium sulfate and concentrated in vacuo. The crude mixture was purified by column chromatography.

2-ethyl-1-(4-(p-tolylthio)butyl)-1H-benzo[d]imidazole

26


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+ Compound 6: Yield = 80%. 1H NMR (300 MHz, Chloroform-d) δ 1.46 (t, J = 7.5 Hz, 3H), 1.55 – 1.78 (m, 2H), 1.78 – 2.05 (m, 2H), 2.32 (s, 3H), 2.69 – 2.99 (m, 4H), 4.07 (t, J = 7.4 Hz, 2H), 7.06 – 7.73 (m, 8H). LCMS – ESI (M+H)+: 325.2. HRMS (ESI): calcd for C20H25N2S+, 325.1733; found, 325.1738. 2-ethyl-1-(4-((4-methoxyphenyl)thio)butyl)-1H-benzo[d]imidazole

Compound 7: Yield = 74%. 1H NMR (300 MHz, Chloroform-d) δ 1.48 (t, J = 7.5 Hz, 3H), 1.54 – 1.72 (m, 2H), 1.82 – 2.08 (m, 2H), 2.70 – 2.98 (m, 4H), 3.81 (s, 3H), 4.10 (t, J = 7.3 Hz, 2H), 6.82 (d, J = 8.6 Hz, 2H), 7.17 – 7.37 (m, 5H), 7.56 – 7.87 (m, 1H). LCMS – ESI (M+H)+: 341.0. HRMS (ESI): calcd for C20H25N2OS+, 341.1682; found, 341.1681. 2-ethyl-1-(4-(naphthalen-2-ylthio)butyl)-1H-benzo[d]imidazole

27



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Page 28 of 64

Compound 8: Yield = 66%. 1H NMR (300 MHz, Chloroform-d) δ 1.45 (t, J = 7.5 Hz, 3H), 1.63 – 1.82 (m, 2H), 1.84 – 2.15 (m, 2H), 2.84 (q, J = 7.5 Hz, 2H), 3.02 (t, J = 6.9 Hz, 2H), 4.08 (t, J = 7.4 Hz, 2H), 7.09 – 7.32 (m, 3H), 7.32 – 7.56 (m, 3H), 7.65 – 7.86 (m, 5H). LCMS – ESI (M+H)+: 361.2. HRMS (ESI): calcd for C23H25N2S+, 361.1733; found, 361.1738. 2-(4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butylthio)quinolone

Compound 9: Yield = 66%. 1H NMR (300 MHz, Chloroform-d) δ 1.44 (t, J = 7.5 Hz, 3H), 1.73 – 2.21 (m, 4H), 2.68 – 3.04 (m, 2H), 3.24 – 3.53 (m, 2H), 4.01 – 4.28 (m, 2H), 7.10 – 7.51 (m, 5H), 7.55 – 7.96 (m, 5H). LCMS – ESI (M+H)+: 362.2. HRMS (ESI): calcd for C22H24N3S+, 362.1685; found, 362.1691. 2-(4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butylthio)quinoxaline

28


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Compound 10: Yield = 78%. 1H NMR (300 MHz, Chloroform-d) δ 1.46 (t, J = 7.5 Hz, 3H), 1.77 – 2.13 (m, 4H), 2.75 – 3.05 (m, 2H), 3.24 – 3.49 (m, 2H), 4.03 – 4.38 (m, 2H), 7.01 – 7.41 (m, 3H), 7.45 – 7.94 (m, 4H), 7.89 – 8.11 (m, 1H), 8.44 – 8.75 (m, 1H). LCMS – ESI (M+H)+: 363.2. HRMS (ESI): calcd for C21H23N4S+, 363.1638; found, 363.1643. 6-ethoxy-2-(4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butylthio)benzo[d]thiazole

Compound 11: Yield = 60%. 1H NMR (300 MHz, Chloroform-d) δ 1.37 – 1.61 (m, 6H), 1.74 – 2.31 (m, 4H), 2.63 – 3.02 (m, 2H), 3.19 – 3.47 (m, 2H), 3.95 – 4.29 (m, 4H), 6.84 – 7.12 (m, 1H), 7.14 – 7.40 (m, 4H), 7.66 – 7.88 (m, 2H). LCMS – ESI (M+H)+: 412.2. HRMS (ESI): calcd for C22H26N3OS2+, 412.1512; found, 412.1517. 6-chloro-2-(4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butylthio)benzo[d]thiazole

29



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Page 30 of 64

Compound 12: Yield = 84%. 1H NMR (300 MHz, Chloroform-d) δ 1.46 (t, J = 7.5 Hz, 3H), 1.80 – 2.12 (m, 4H), 2.74 – 3.01 (m, 2H), 3.24 – 3.48 (m, 2H), 3.96 – 4.26 (m, 2H), 7.11 – 7.54 (m, 4H), 7.63 – 7.96 (m, 3H). LCMS – ESI (M+H)+: 402.1. HRMS (ESI): calcd for C20H21ClN3S2+, 402.0860; found, 402.0865. 1-(4-(1H-benzo[d]imidazol-2-ylthio)butyl)-2-ethyl-1H-benzo[d]imidazole

Compound 13: Yield = 77%. 1H NMR (300 MHz, CDCl3): δ 1.47 (t, J = 7.5 Hz, 3H); 1.93 (m, 4H); 2.85 (q, J = 7.5 Hz, 2H); 3.32 (t, J = 5.4 Hz, 2H); 4.02 (t, J = 5.7 Hz, 2H); 7.15 – 8.02 (m, 8H). LCMS – ESI (M+H)+: 351.2. HRMS (ESI): calcd for C20H23N4S+, 351.1638; found, 351.1643. 2-ethyl-1-(4-((1-methyl-1H-benzo[d]imidazol-2-yl)thio)butyl)-1H-benzo[d]imidazole

30


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Compound 14: Yield = 99%. 1H NMR (300 MHz, Chloroform-d) δ 1.45 (t, J = 7.5 Hz, 3H), 1.78 – 2.06 (m, 4H), 2.79 – 2.91 (m, 2H), 3.29 – 3.49 (m, 2H), 3.65 (s, 3H), 3.98 – 4.24 (m, 2H), 7.09 – 7.38 (m, 6H), 7.54 – 7.82 (m, 2H). LCMS – ESI (M+H)+: 365.2. HRMS (ESI): calcd for C21H25N4S+, 365.1794; found, 365.1800. 2-(4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butylthio)thiazole

Compound 15: Yield = 79%. 1H NMR (300 MHz, Chloroform-d) δ 1.47 (t, J = 7.4 Hz, 3H), 1.75 – 2.11 (m, 4H), 2.59 – 3.06 (m, 2H), 3.12 – 3.38 (m, 2H), 3.97 – 4.23 (m, 2H), 7.13 – 7.35 (m, 4H), 7.58 – 7.79 (m, 2H). LCMS – ESI (M+H)+: 318.1. HRMS (ESI): calcd for C16H20N3S2+, 318.1093; found, 318.1099. 2-(4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butylthio)-5-methylthiazole N N

N S S

Compound 16: Yield 50%. 1H NMR (300 MHz, Chloroform-d) δ 1.47 (t, J = 7.5 Hz, 3H), 1.74 – 2.04 (m, 4H), 2.72 (s, 3H), 2.88 (q, J = 7.5 Hz, 2H), 3.32 (t, J = 6.8 Hz, 2H), 4.13 (t, J = 7.0 Hz, 2H), 7.18 – 7.38 (m, 4H), 7.65 – 7.77 (m, 1H). LCMS – ESI (M+H)+: 332.9. HRMS (ESI): calcd for C17H22N3S2+, 332.1250; found, 332.1255. 2-ethyl-1-(4-((1-methyl-1H-tetrazol-5-yl)thio)butyl)-1H-benzo[d]imidazole

31



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Compound 17: Yield = 95%. 1H NMR (300 MHz, Chloroform-d) δ 1.47 (t, J = 7.5 Hz, 3H), 1.78 – 2.06 (m, 4H), 2.77 – 3.01 (m, 2H), 3.33 (t, J = 6.7 Hz, 2H), 3.86 (s, 3H), 4.13 (t, J = 6 Hz, 2H), 7.25 (m, 3H), 7.64 – 7.83 (m, 1H). LCMS – ESI (M+H)+: 317.2. HRMS (ESI): calcd for C15H21N6S+, 317.1543; found, 317.1548. 2-((4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butyl)thio)-4-phenylthiazole

Compound 18: Yield = 90%. 1H NMR (300 MHz, CDCl3): 1.46 (t, J = 7.5 Hz, 3H); 1.94 (m, 4H); 2.88 (q, J = 7.2 Hz, 2H); 3.28 (t, J = 7 Hz, 2H); 4.10 (t, J = 7 Hz, 2H); 7.19 – 7.64 (m, 10H). LCMS – ESI (M+H)+: 394.2. HRMS (ESI): calcd for C22H24N3S2+, 394.1406; found, 394.1412. 2-(4-chlorophenyl)-5-((3-(2-ethyl-1H-benzo[d]imidazol-1-yl)propyl)thio)-1,3,4-oxadiazole

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Compound 20: Yield = 50%. 1H NMR (300 MHz, CDCl3): δ 1.49 (t, J = 7.5 Hz, 3H); 2.41 (m, 2H); 2.94 (q, J = 6.8 Hz, 2H); 3.30 (t, J = 6.9 Hz, 2H); 4.31 (t, J = 7.2 Hz, 2H); 7.22 – 7.94 (m, 8H). LCMS – ESI (M+H)+: 399.2. HRMS (ESI): calcd for C20H20ClN4OS+, 399.1041; found, 399.1047. 2-(4-chlorophenyl)-5-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)ethyl)thio)-1,3,4-oxadiazole

Compound 21: Yield = 71%. 1H NMR (300 MHz, Chloroform-d) δ 1.50 (t, J = 7.5 Hz, 3H), 2.97 (q, J = 7.5 Hz, 2H), 3.60 (t, J = 6.9 Hz, 2H), 4.66 (t, J = 7.2 Hz, 2H), 7.18 – 7.33 (m, 2H), 7.44 – 7.55 (m, 4H), 7.68 – 7.80 (m, 1H), 7.94 (d, J = 8.6 Hz, 2H). LCMS – ESI (M+H)+: 385.10. HRMS (ESI): calcd for C19H18ClN4OS+, 385.0884; found, 385.0894. 2-(4-chlorophenyl)-5-((3-(2-ethyl-6-methyl-1H-benzo[d]imidazol-1-yl)propyl)thio)-1,3,4oxadiazole

Compound 22: Yield = 38%. 1H NMR (300 MHz, CDCl3): δ 1.45 (t, J = 7.5 Hz, 3H); 2.47 (m, 2H); 2.88 (q, J = 7.0 Hz, 2H); 3.29 (t, J = 6.6 Hz, 2H); 4.28 (t, J = 7.5 Hz, 2H); 7.04 – 7.94 (m, 7H). LCMS – ESI (M+H)+: 413.1. HRMS (ESI): calcd for C21H22ClN4OS+, 413.1197; found, 413.1203. 33



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2-(4-chlorophenyl)-5-((4-(2-ethyl-6-methyl-1H-benzo[d]imidazol-1-yl)butyl)thio)-1,3,4oxadiazole

Compound 23: Yield = 31%. 1H NMR (300 MHz, CDCl3): δ 1.45 (t, J = 7.5 Hz, 3H); 1.98 (m, 4H); 2.47 (s, 3H); 2.88 (q, J = 6.7 Hz, 2H); 3.30 (t, J = 7.0 Hz, 2H); 4.13 (t, J = 7.5 Hz, 2H); 7.03 – 7.95 (m, 7H). LCMS – ESI (M+H)+: 427.1. HRMS (ESI): calcd for C22H24ClN4OS+, 427.1354; found, 427.1359. 2-(4-chlorophenyl)-5-((3-(2-ethyl-6-methoxy-1H-benzo[d]imidazol-1-yl)propyl)thio)-1,3,4oxadiazole

Compound 24: Yield = 63%. 1H NMR (300 MHz, Chloroform-d) δ 1.48 (t, J = 7.5 Hz, 3H), 2.32 – 2.54 (m, 2H), 2.83 – 2.94 (m, 2H), 3.30 (t, J = 7.0 Hz, 2H), 3.87 (s, 3H), 4.26 (t, J = 7.1 Hz, 2H), 6.73 – 6.93 (m, 2H), 7.49 (d, J = 8.6 Hz, 2H), 7.62 (d, J = 8.7 Hz, 1H), 7.93 (d, J = 8.6 Hz, 2H). LCMS – ESI (M+H)+: 428.9. HRMS (ESI): calcd for C21H22ClN4O2S+, 429.1147; found, 429.1155. N-(4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butyl)-4-methylaniline

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Compound 25: Yield = 27%. 1H NMR (300 MHz, Chloroform-d) δ 1.47 (t, J = 7.3 Hz, 3H), 1.60 – 1.80 (m, 2H), 1.81 – 1.99 (m, 2H), 2.24 (s, 3H), 2.89 (q, J = 7.3 Hz, 2H), 3.13 (t, J = 6.6 Hz, 2H), 3.44 (broad s, 1H), 4.14 (t, J = 7.1 Hz, 2H), 6.51 (d, J = 8.0 Hz, 2H), 6.99 (d, J = 7.7 Hz, 2H), 7.14 – 7.37 (m, 3H), 7.64 – 7.87 (m, 1H). LCMS – ESI (M+H)+: 308.2. HRMS (ESI): calcd for C20H26N3+, 308.2121; found, 308.2119. N-(4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butyl)-4-methoxyaniline

Compound 26: Yield = 82%. 1H NMR (300 MHz, Chloroform-d) δ 1.49 (t, J = 7.5 Hz, 3H), 1.57 – 1.76 (m, 2H), 1.84 – 2.07 (m, 2H), 2.90 (q, J = 7.5 Hz, 2H), 3.11 (t, J = 6.9 Hz, 2H), 3.76 (s, 4H), 4.15 (t, J = 7.3 Hz, 2H), 6.57 (d, J = 8.8 Hz, 2H), 6.80 (d, J = 8.8 Hz, 2H), 7.09 – 7.35 (m, 3H), 7.63 – 7.82 (m, 1H). LCMS – ESI (M+H)+: 324.0. HRMS (ESI): calcd for C20H26N3O+, 324.2070; found, 324.2081. N-(4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butyl)-3-(trifluoromethyl)aniline

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Compound 27: Yield = 53%. 1H NMR (300 MHz, Chloroform-d) δ 1.50 (t, J = 7.4 Hz, 3H), 1.57 – 1.80 (m, 2H), 1.80 – 2.10 (m, 2H), 2.91 (q, J = 7.2 Hz, 2H), 3.00 – 3.28 (m, 2H), 3.80 (broad s, 1H), 4.00 – 4.28 (m, 2H), 6.50 – 6.84 (m, 2H), 6.80 – 7.05 (m, 1H), 7.04 – 7.43 (m, 4H), 7.60 – 7.85 (m, 1H). LCMS – ESI (M+H)+: 362.2. HRMS (ESI): calcd for C20H23F3N3+, 362.1839; found, 362.1834. N-(4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butyl)-3,5-dimethoxyaniline

Compound 28: Yield =73%. 1H NMR (300 MHz, Chloroform-d) δ 1.49 (t, J = 7.4 Hz, 3H), 1.57 – 1.76 (m, 2H), 1.79 – 1.99 (m, 2H), 2.80 – 2.95 (m, 2H), 3.13 (t, J = 7.1 Hz, 2H), 3.76 (broad s, 6 H), 4.14 (t, J = 7.1 Hz, 2H), 5.68 – 5.99 (m, 3H), 7.07 – 7.35 (m, 3H), 7.66 – 7.87 (m, 1H). LCMS – ESI (M+H)+: 354.2. HRMS (ESI): calcd for C21H28N3O2+, 354.2176; found, 354.2156. 3-bromo-N-(4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butyl)-4-methylaniline

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Compound 29: Yield = 56%. 1H NMR (300 MHz, Chloroform-d) δ 1.49 (t, J = 7.5 Hz, 3H), 1.79 – 2.09 (m, 4H), 2.90 (q, J = 7.9 Hz, 2H), 3.41 (t, J = 6.8 Hz, 2H), 3.74 (broad s, 1H), 4.14 (t, J = 6.7 Hz, 2H), 7.03 – 7.38 (m, 5H), 7.38 – 7.89 (m, 2H). LCMS – ESI (M+H)+: 386.1. HRMS (ESI): calcd for C20H25BrN3+, 386.1226; found, 386.1224. 3-chloro-N-(4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butyl)-4-methylaniline

Compound 30: Yield = 51%. 1H NMR (300 MHz, Chloroform-d) δ 1.50 (t, J = 7.5 Hz, 3H), 1.59 – 1.78 (m, 2H), 1.78 – 2.00 (m, 2H), 2.26 (s, 3H), 2.90 (q, J = 7.5 Hz, 2H), 3.12 (t, J = 6.9 Hz, 2H), 3.53 (broad s, 1H), 4.15 (t, J = 7.3 Hz, 2H), 6.40 (d, J = 8.4 Hz, 1H), 6.60 (d, J = 2.5 Hz, 1H), 7.00 (d, J = 8.2 Hz, 1H), 7.17 – 7.36 (m, 3H), 7.58 – 7.84 (m, 1H). LCMS – ESI (M+H)+: 342.0. HRMS (ESI): calcd for C20H25ClN3+, 342.1732; found, 342.1733. 3-(benzyloxy)-N-(4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butyl)aniline

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Compound 31: Yield = 48%. 1H NMR (300 MHz, Chloroform-d) δ 1.46 (t, J = 7.5 Hz, 3H), 1.53 – 1.71 (m, 2H), 1.73 – 1.96 (m, 2H), 2.86 (q, J = 7.5 Hz, 2H), 3.10 (t, J = 6.9 Hz, 2H), 3.61 (broad s, 1H), 4.10 (t, J = 7.3 Hz, 2H), 5.01 (s, 2H), 6.08 – 6.26 (m, 2H), 6.28 – 6.40 (m, 1H), 6.93 – 7.12 (m, 1H), 7.18 – 7.49 (m, 7H), 7.65 – 7.80 (m, 1H). LCMS – ESI (M+H)+: 400.2. HRMS (ESI): calcd for C26H30N3O+, 400.2383; found, 400.2389. N-(4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butyl)-4-(pyridin-2-yl)aniline

Compound 32: Yield = 42%. 1H NMR (300 MHz, Chloroform-d) δ 1.47 (t, J = 7.5 Hz, 3H), 1.59 – 1.80 (m, 2H), 1.82 – 2.06 (m, 2H), 2.88 (q, J = 7.5 Hz, 2H), 3.09 – 3.30 (m, 3H), 4.07 – 4.24 (m, 2H), 6.58 – 6.71 (m, 2H), 7.03 – 7.16 (m, 1H), 7.17 – 7.34 (m, 3H), 7.57 – 7.78 (m, 3H), 7.79 – 7.90 (m, 2H), 8.53 – 8.66 (m, 1H). LCMS – ESI (M+H)+: 371.2. HRMS (ESI): calcd for C24H27N4+, 371.2230; found, 371.2229. 38


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N-(4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butyl)-3-(pyridin-2-yl)aniline

Compound 33: Yield = 50%. 1H NMR (300 MHz, Chloroform-d) δ 1.49 (t, J = 7.5 Hz, 3H), 1.59 – 1.82 (m, 2H), 1.79 – 2.10 (m, 2H), 2.91 (q, J = 8.1 Hz, 2H), 3.10 – 3.38 (m, 2H), 3.74 (broad s, 1H), 3.99 – 4.28 (m, 2H), 6.48 – 6.75 (m, 1H), 7.04 – 7.42 (m, 7H), 7.48 – 7.84 (m, 3H), 8.53 – 8.82 (m, 1H). LCMS – ESI (M+H)+: 371.2. HRMS (ESI): calcd for C24H27N4+, 371.2230; found, 371.2219. N-benzyl-4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butan-1-amine

Compound 34: Yield = 81%. 1H NMR (300 MHz, Chloroform-d) δ 1.46 (t, J = 7.5 Hz, 3H), 1.59 – 2.03 (m, 4H), 2.88 (q, J = 7.5 Hz, 2H), 3.50 (t, J = 6.9 Hz, 2H), 4.13 (t, J = 7.0 Hz, 2H), 4.49 (s, 2H), 7.09 – 7.43 (m, 8H), 7.54 – 7.84 (m, 1H). LCMS – ESI (M+H)+: 308.2. HRMS (ESI): calcd for C20H26N3+, 308.2121; found, 308.2127. 4-(4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butyl)morpholine 39



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Compound 35: Yield = 64%. 1H NMR (300 MHz, Chloroform-d) δ 1.42 (t, J = 7.5 Hz, 3H), 1.46 – 1.66 (m, 2H), 1.72 – 1.94 (m, 2H), 2.23 – 2.56 (m, 6H), 2.95 (q, J = 7.5 Hz, 2H), 3.53 – 3.72 (m, 4H), 4.24 (t, J = 7.4 Hz, 2H), 7.17 – 7.33 (m, 2H), 7.47 (d, J = 7.6 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H). LCMS – ESI (M+H)+: 288.2. HRMS (ESI): calcd for C17H26N3O+, 288.2070; found, 288.2073. N-(3-(2-ethyl-1H-benzo[d]imidazol-1-yl)propyl)aniline

Compound 37: Yield = 29%. 1H NMR (300 MHz, Chloroform-d) δ 1.45 (t, J = 7.5 Hz, 3H), 2.13 (m, 2H), 2.88 (q, J = 7.5 Hz, 2H), 3.18 (t, J = 6.5 Hz, 2H), 3.59 (broad s, 1H), 4.26 (t, J = 7.0 Hz, 2H), 6.58 (d, J = 8.5 Hz, 2H), 6.74 (t, J = 7.3 Hz, 1H), 6.99 – 7.37 (m, 5H), 7.63 – 7.86 (m, 1H). LCMS – ESI (M+H)+: 280.2. HRMS (ESI): calcd for C18H22N3+, 280.1808; found, 280.1814. 2-ethyl-1-(3-(p-tolyloxy)propyl)-1H-benzo[d]imidazole

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Compound 38: Yield = 80%. 1H NMR (300 MHz, Chloroform-d) δ 1.43 (t, J = 7.5 Hz, 3H), 2.18 – 2.37 (m, 2H), 2.88 (q, J = 7.4 Hz, 2H), 3.88 (t, J = 6.5 Hz, 2H), 4.35 (t, J = 6.6 Hz, 2H), 6.78 (d, J = 8.3 Hz, 2H), 7.08 (d, J = 8.0 Hz, 2H), 7.13 – 7.39 (m, 3H), 7.73 (m, 1H). LCMS – ESI (M+H)+: 295.2. HRMS (ESI): calcd for C19H23ON2+, 295.1805; found, 295.1803. 2-ethyl-1-(3-(p-tolylthio)propyl)-1H-benzo[d]imidazole

Compound 39: Yield = 54%. 1H NMR (300 MHz, Chloroform-d) δ 1.44 (t, J = 7.5 Hz, 3H), 1.87 – 2.21 (m, 2H), 2.32 (s, 3H), 2.74 – 2.99 (m, 4H), 4.09 – 4.38 (m, 2H), 7.01 – 7.38 (m, 7H), 7.61 – 7.82 (m, 1H). LCMS – ESI (M+H)+: 311.1. HRMS (ESI): calcd for C19H23N2S+, 311.1576; found, 311.1574. N-(3-(2-ethyl-1H-benzo[d]imidazol-1-yl)propyl)-4-methylaniline

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Compound 40: Yield = 48%. 1H NMR (300 MHz, Chloroform-d) δ 1.45 (t, J = 7.4 Hz, 3H), 2.11 (m, 2H), 2.24 (s, 3H), 2.87 (q, J = 7.4 Hz, 2H), 3.14 (t, J = 6.8 Hz, 2H), 3.47 (broad s, 1H), 4.24 (t, J = 6.9 Hz, 2H), 6.51 (d, J = 8.7 Hz, 2H), 6.99 (d, J = 7.7 Hz, 2H), 7.11 – 7.38 (m, 3H), 7.60 – 7.84 (m, 1H). LCMS – ESI (M+H)+: 294.2. HRMS (ESI): calcd for C19H24N3+, 294.1965; found, 294.1969. 1-(3-(3,4-dimethylphenoxy)propyl)-2-ethyl-1H-benzo[d]imidazole

Compound 41: Yield = 74%. 1H NMR (300 MHz, Chloroform-d) δ 1.43 (t, J = 7.5 Hz, 3H), 2.03 – 2.38 (m, 8H), 2.88 (q, J = 7.4 Hz, 2H), 3.88 (t, J = 6.2 Hz, 2H), 4.34 (t, J = 6.7 Hz, 2H), 6.54 – 6.66 (m, 1H), 6.69 (s, 1H), 7.03 (d, J = 8.2 Hz, 1H), 7.12 – 7.27 (m, 2H), 7.30 – 7.41 (m, 1H), 7.73 (d, J = 7.1 Hz, 1H). LCMS – ESI (M+H)+: 309.3. HRMS (ESI): calcd for C20H25N2O+, 309.1961; found, 309.1957. 3-bromo-N-(3-(2-ethyl-1H-benzo[d]imidazol-1-yl)propyl)-4-methylaniline

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Compound 42: Yield = 26%. 1H NMR (300 MHz, Chloroform-d) δ 1.46 (t, J = 7.5 Hz, 3H), 1.97 – 2.21 (m, 2H), 2.28 (s, 3H), 2.87 (q, J = 7.2 Hz, 2H), 3.12 (t, J = 6.4 Hz, 2H), 4.24 (t, J = 6.5 Hz, 2H), 6.42 (d, J = 8.2 Hz, 1H), 6.79 (s, 1H), 7.00 (d, J = 8.1 Hz, 1H), 7.15 – 7.44 (m, 3H), 7.75 (d, J = 7.2 Hz, 1H). LCMS – ESI (M+H)+: 371.9. HRMS (ESI): calcd for C19H23BrN3+, 372.1070; found, 372.1055. 1-(3-(3-chloro-4-methylphenoxy)propyl)-2-ethyl-1H-benzo[d]imidazole

Compound 43: Yield = 80%. 1H NMR (300 MHz, Chloroform-d) δ 1.43 (t, J = 7.5 Hz, 3H), 2.18 – 2.28 (m, 2H), 2.30 (s, 3H), 2.87 (q, J = 7.5 Hz, 2H), 3.86 (t, J = 6.5 Hz, 2H), 4.33 (t, J = 6.8 Hz, 2H), 6.69 (d, J = 8.4 Hz, 1H), 6.89 (d, J = 2.3 Hz, 1H), 7.11 (d, J = 8.5 Hz, 1H), 7.18 – 7.36 (m, 3H), 7.74 (d, J = 8.2 Hz, 1H). LCMS – ESI (M+H)+: 329.2. HRMS (ESI): calcd for C19H22ClN2O+, 329.1415; found, 329.1406. 2-ethyl-6-methyl-1-(4-(phenylthio)butyl)-1H-benzo[d]imidazole

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Compound 45: Yield = 21%. 1H NMR (300 MHz, Chloroform-d) δ 1.45 (t, J = 7.5 Hz, 3H), 1.56 – 1.83 (m, 2H), 1.84 – 2.13 (m, 2H), 2.48 (s, 3H), 2.74 – 3.06 (m, 4H), 3.92 – 4.23 (m, 2H), 6.96 – 7.40 (m, 6H), 7.45 – 7.72 (m, 1H). LCMS – ESI (M+H)+: 325.2. 2-ethyl-1-(4-(4-methoxyphenoxy)butyl)-6-methyl-1H-benzo[d]imidazole

Compound 47: Yield = 70%. 1H NMR (300 MHz, Chloroform-d) δ 1.48 (t, J = 7.5 Hz, 3H), 1.72 – 1.93 (m, 2H), 1.92 – 2.11 (m, 2H), 2.49 (s, 3H), 2.90 (q, J = 7.5 Hz, 2H), 3.78 (s, 3H), 3.86 – 4.00 (m, 2H), 4.10 – 4.27 (m, 2H), 6.73 – 6.96 (m, 4H), 6.99 – 7.25 (m, 2H), 7.47 – 7.69 (m, 1H). LCMS – ESI (M+H)+: 339.0. HRMS (ESI): calcd for C21H27N2O2+, 339.2067; found, 339.2064. N-(4-(2-ethyl-6-methyl-1H-benzo[d]imidazol-1-yl)butyl)-4-methoxyaniline

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Compound 48: Yield = 70%. 1H NMR (300 MHz, Chloroform-d) δ 1.46 (t, J = 7.4 Hz, 3H), 1.59 – 1.80 (m, 2H), 1.81 – 2.01 (m, 2H), 2.49 (s, 3H), 2.78 – 2.94 (m, 3H), 3.11 (q, J = 6.5 Hz, 2H), 3.76 (s, 3H), 3.99 – 4.26 (m, 2H), 6.37 – 6.68 (m, 2H), 6.79 (d, J = 7.8 Hz, 2H), 7.06 (d, J = 8.0 Hz, 2H), 7.48 – 7.75 (m, 1H). LCMS – ESI (M+H)+: 338.1. HRMS (ESI): calcd for C21H28N3O+, 338.2227; found, 338.2221. 2-ethyl-1-(4-((4-methoxyphenyl)thio)butyl)-6-methyl-1H-benzo[d]imidazole

Compound 49: Yield = 80%. 1H NMR (300 MHz, Chloroform-d) δ 1.46 (t, J = 7.5 Hz, 3H), 1.52 – 1.73 (m, 2H), 1.83 – 2.02 (m, 2H), 2.50 (s, 3H), 2.75 – 2.93 (m, 4H), 3.80 (s, 3H), 3.94 – 4.17 (m, 2H), 6.58 – 6.90 (m, 2H), 6.98 – 7.22 (m, 2H), 7.22 – 7.38 (m, 2H), 7.46 – 7.69 (m, 1H). LCMS – ESI (M+H)+: 355.0. HRMS (ESI): calcd for C21H27N2OS+, 355.1839; found, 355.1835. 3-chloro-N-(4-(2-ethyl-6-methyl-1H-benzo[d]imidazol-1-yl)butyl)aniline

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Compound 50: Yield = 15%. 1H NMR (300 MHz, CDCl3): δ 1.45 (t, J = 7.5 Hz, 3H); 1.62 (m, 2H); 1.90 (m, 2H); 2.49 (s, 3H); 2.86 (q, J = 7.5 Hz, 2H); 3.40 (broad s, 1H); 3.54 (t, J = 6.9 Hz, 2H); 4.13 (t, J = 6.7 Hz, 2H); 7.06 – 7.62 (m, 7H). LCMS – ESI (M+H)+: 342.0. N-(4-(2-ethyl-6-methyl-1H-benzo[d]imidazol-1-yl)butyl)-3-(trifluoromethyl)aniline

Compound 51: Yield = 27%. 1H NMR (300 MHz, CDCl3): δ 1.45 (t, J = 7.5 Hz, 3H); 1.66 (m, 2H); 1.90 (m, 2H); 2.47 (s, 3H); 2.86 (q, J = 7.5 Hz, 2H); 3.08 (t, J = 6.9 Hz, 2H); 3.70 (broad s, 1H); 4.13 (t, J = 5.7 Hz, 2H); 6.68 – 8.01 (m, 7H). LCMS – ESI (M+H)+: 376.2. HRMS (ESI): calcd for C21H25F3N3+, 376.1995; found, 376.1979. 3-(benzyloxy)-N-(4-(2-ethyl-6-methyl-1H-benzo[d]imidazol-1-yl)butyl)aniline

46


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Compound 52: Yield = 43%. 1H NMR (300 MHz, Chloroform-d) δ 1.45 (t, J = 7.2 Hz, 3H), 1.53 – 1.99 (m, 4H), 2.48 (s, 3H), 2.74 – 2.92 (m, 2H), 3.12 (q, J = 6.4 Hz, 2H), 3.61 (broad s, 1H), 3.99 – 4.19 (m, 2H), 5.02 (s, 2H), 5.99 – 6.30 (m, 2H), 6.35 (d, J = 8.6 Hz, 1H), 6.95 – 7.68 (m, 9H). LCMS – ESI (M+H)+: 414.3. HRMS (ESI): calcd for C27H32N3O+, 414.2540; found, 414.2531. 1-(4-(4-chlorophenylthio)butyl)-2-ethyl-6-methyl-1H-benzo[d]imidazole

Compound 53: Yield = 89%. 1H NMR (300 MHz, Chloroform-d) δ 1.44 (t, J = 7.5 Hz, 3H), 1.57 – 1.77 (m, 2H), 1.84 – 2.04 (m, 2H), 2.48 (s, 3H), 2.66 – 2.98 (m, 4H), 4.05 (t, J = 8.8 Hz, 2H), 6.99 – 7.31 (m, 6H), 7.46 – 7.68 (m, 1H). LCMS – ESI (M+H)+: 359.1. HRMS (ESI): calcd for C20H24ClN2S+, 359.1343; found, 359.1349. N-(3-(2-ethyl-6-methyl-1H-benzo[d]imidazol-1-yl)propyl)aniline

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Compound 54 : Yield = 61%. 1H NMR (300 MHz, Chloroform-d) δ 1.46 (t, J = 7.5 Hz, 3H), 1.98 – 2.26 (m, 2H), 2.45 (s, 3H), 2.87 (q, J = 7.5 Hz, 2H), 3.19 (t, J = 6.1 Hz, 2H), 3.64 (s, 1H), 4.23 (t, J = 7.0 Hz, 2H), 6.60 (d, J = 7.7 Hz, 2H), 6.76 (t, J = 7.3 Hz, 1H), 6.98 – 7.13 (m, 2H), 7.21 (t, J = 7.9 Hz, 2H), 7.63 (d, J = 8.6 Hz, 1H). LC-MS-ESI (M + H)+, 294.1 N-(3-(2-ethyl-6-methyl-1H-benzo[d]imidazol-1-yl)propyl)-4-methylaniline

Compound 55: Yield = 23%. 1H NMR (300 MHz, Chloroform-d) δ 1.44 (t, J = 7.5 Hz, 3H), 1.90 – 2.20 (m, 2H), 2.28 (s, 3H), 2.47 (s, 3H), 2.85 (q, J = 7.5 Hz, 2H), 3.01 – 3.22 (m, 2H), 4.06 – 4.30 (m, 2H), 6.28 – 6.51 (m, 1H), 6.67 – 6.79 (m, 1H), 6.96 – 7.25 (m, 3H), 7.45 – 7.70 (m, 2H). LCMS – ESI (M+H)+: 308.2. N-(3-(2-ethyl-6-methyl-1H-benzo[d]imidazol-1-yl)propyl)-4-methoxyaniline

48


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Compound 56: Yield = 42%. 1H NMR (300 MHz, CDCl3): δ 1.44 (t, J = 7.5 Hz, 3H); 2.04 (m, 2H); 2.47 (s, 3H); 2.91 (m, 4H); 3.81 (s, 3H); 4.22 (t, J = 7.2 Hz, 2H); 6.84 – 7.61 (m, 7H). LCMS – ESI (M+H)+: 324.1. HRMS (ESI): calcd for C20H26N3O+, 324.2070; found, 324.2084. N-(3-(2-ethyl-6-methyl-1H-benzo[d]imidazol-1-yl)propyl)-2,4-dimethylaniline

Compound 57: Yield = 28%. 1H NMR (300 MHz, CDCl3): δ 1.45 (t, J = 2.4 Hz, 3H); 2.19 (m, 8H); 2.47 (s, 3H); 2.88 (q, J = 6.7 Hz, 2H); 3.17 (t, J = 6.6 Hz, 2H); 3.50 (s, 1H); 4.23 (t, J = 7.2 Hz, 2H); 6.41 – 7.61 (m, 6H). LCMS – ESI (M+H)+: 322.2. HRMS (ESI): calcd for C21H28N3+, 322.2283; found, 322.2276. 2-ethyl-1-(3-((4-methoxyphenyl)thio)propyl)-6-methyl-1H-benzo[d]imidazole N N

S

O

Compound 58: Yield = 62%. 1H NMR (300 MHz, Chloroform-d) δ 1.44 (t, J = 7.5 Hz, 3H), 1.94 – 2.15 (m, 2H), 2.47 (s, 3H), 2.71 – 2.97 (m, 4H), 3.81 (s, 3H), 4.21 (t, J = 8.3 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H), 7.00 – 7.24 (m, 2H), 7.33 – 7.43 (m, 2H), 7.51 – 7.70 (m, 1H). LCMS – ESI (M+H)+: 341.0. HRMS (ESI): calcd for C20H25N2OS+, 341.1682; found, 341.1677. 1-(4-phenoxybutyl)-1H-imidazole 49



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Compound 59: LCMS – ESI (M+H)+: 217.0, tR = 0.759 min, purity >99 %. 1-(4-(4-chlorophenoxy)butyl)-1H-imidazole

Compound 60: LCMS – ESI (M+H)+: 250.9, tR = 0.688 min, purity 92 %. 1-(4-(2,4-dichlorophenoxy)butyl)-1H-imidazole

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Compound 61: LCMS – ESI (M+H)+: 284.8, tR = 0.662 min, purity 85 %. 1-(4-(4-bromo-2-chlorophenoxy)butyl)-1H-imidazole

Compound 62: LCMS – ESI (M+H)+: 330.7, tR = 4.032 min, purity 95 %. 1-(4-(2-chloro-4-methylphenoxy)butyl)-1H-imidazole

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Compound 63: LCMS – ESI (M+H)+: 264.9, tR = 0.667 min, purity >99%. 1-(4-(4-chlorophenoxy)butyl)-2-methyl-1H-imidazole

Compound 64: LCMS – ESI (M+H)+: 264.9, tR = 3.716 min, purity 95%. 1-(4-(4-chlorophenylthio)butyl)-1H-imidazole

Compound 65: LCMS – ESI (M+H)+: 266.9, tR = 3.559 min, purity >99 %. 1-(6-(2,4-dichlorophenoxy)hexyl)-1H-imidazole

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Compound 66: LCMS – ESI (M+H)+: 312.8 , tR = 4.441 min, purity 95%. 1-(3-(naphthalen-2-yloxy)propyl)-1H-imidazole

Compound 67: LCMS – ESI (M+H)+: 252.9, tR = 3.667 min, purity >99 %.

1-(6-(2,4-dimethylphenoxy)hexyl)-1H-imidazole

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Compound 68: LCMS – ESI (M+H)+: 273.0, tR = 4.391 min, purity 95 %.

AUTHOR CONTRIBUTION NSC, BB, GS, TOM, AM, LF, and TI conducted experimental work. NSC, BB, GS, SC, TOM, AM, LF, TI, JS, TM, PAH, JO, and TP conceived and designed the experiments and analyzed the data. NSC, BB, GS, SC, JO, and TP wrote the paper.

FUNDING Research reported in this publication was supported by NIAID of the National Institutes of Health under award number R21AI107626 and the Bill & Melinda Gates Foundation, under grant number OPP1024038 and OPP1024055.

ACKNOWLEDGEMENTS 54


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We thank Alfredo Blakeley, James Ahn, Mai Ann Bailey, Jack Elder, Megan Files, Stephanie Florio, Julianne Olinger, Aaron Korkegian and Yulia Ovechkina for technical assistance.

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Figure 1

PAB compounds deplete ATP and disrupt pH homeostasis. (A) ATP levels of M. tuberculosis H37Rv-LP treated with compounds at the indicated concentrations for 24 hrs. ATP levels are calculated as relative luminescence units divided by optical density to account for potential differences in bacterial growth. (B) Intrabacterial pH of M. tuberculosis treated with PAB or reference compounds for 48 hrs. Compound # refers to compounds as listed in tables above. Data are representative of at least two independent experiments.

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Figure 2

PAB compounds are bactericidal against nutrient-starved M. tuberculosis. PAB compounds at 10X their respective MICs were added to M. tuberculosis grown aerobically (A) or after nutrient starvation in PBS 0.05% Tyloxapol for 2 weeks (B). Serial dilutions were performed and plated for CFUs at the indicated times. Data are representative of at least two independent experiments.

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For Table of Contents Use Only

N

Wild type MIC90

0.056 µM

QcrBM342T MIC90

4.1 µM

QcrBT313I MIC90

8.7 µM

Intracellular IC90

0.028 µM

Raw Cytotoxicity TC50

> 50 µM

N N H

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N N

N

Wild type QcrBM342T Rv1339V90M Intracellular Cytotoxicity

MIC = 5.7 MIC >100 MIC = 9.6 MIC90 = 0.86 TC50 =17

S S


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Improved Phenoxyalkylbenzimidazoles with Activity against

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